To determine whether apoptosis of endothelial and connective tissue cells is responsible for the loss of cellularity observed in implanted aortic allograft valves, fresh (n=6) and cryopreserved (n=4) aortic allograft valves were retrieved at 2 days - 20 weeks after implantation in an ovine model. Sections of these valves were studied using histological and electron microscopic methods, nick end labeling and dual immunostaining for Factor VIII-related antigen and proliferating cell nuclear antigen (PCNA), followed by counterstaining for DNA (DAPI) and laser scanning confocal fluorescence microscopic observation. The endothelial cells and leaflet connective tissue cells of implanted valvular allografts showed loss of PCNA (indicative of cessation of mitotic activity), and evidence of apoptosis (nick end labeling). The latter was manifested by nuclear condensation and pyknosis, positive nick end labeling and formation of intra- and extracellular apoptotic bodies derived from the fragmentation of apoptotic cells. These changes began to develop at 2 days after implantation, peaking at 10-14 days, and became complete by 20 weeks, at which time the valves had the typical acellular morphology of allografts implanted for long periods of time. Apoptosis occurs in endothelial cells and leaflet connective tissue cells of implanted allografts and appears to be a major cause of their loss of cellularity. This apoptosis may be related to various factors, including immunologic and chemical injury, and hypoxia during valve processing and reperfusion injury at the time of implantation.